Locke Gregory A, Cheng Dong, Witmer Mark R, Tamura James K, Haque Tasir, Carney Robert F, Rendina Alan R, Marcinkeviciene Jovita
Department of Chemical Enzymology, Bristol Myers-Squibb Pharmaceutical Company, R&D, P.O. Box 5400, Princeton, NJ 08543-5400, USA.
Arch Biochem Biophys. 2008 Jul 1;475(1):72-9. doi: 10.1016/j.abb.2008.04.011. Epub 2008 Apr 18.
The role of citrate as a physiological modulator of mammalian acetyl-CoA carboxylases (ACCs) has been well studied; however, the mechanism has not been clearly defined. In the current study, we found that citrate activated recombinant human ACC2 by more than approximately 1000-fold, but activated recombinant human ACC1 only by approximately 4-fold. The data fit best to a model which accounts for cooperative binding of two citrate molecules. Citrate activates ACCs at lower concentrations and inhibits at higher concentrations with apparent K(d) values of 0.8+/-0.3 and 3.4+/-0.6 mM, and apparent K(i) values of 20+/-8 and 38 +/-8 mM for ACC1 and ACC2, respectively. In the absence of added citrate, both ACC1 and ACC2 were inactivated by avidin rapidly and completely. Addition of 10 mM citrate protected ACC2 from avidin inactivation; however, protection by citrate was less pronounced for ACC1. In response to citrate treatment, different aggregation patterns for the two isoforms were also observed by dynamic light scattering. Although formation of aggregates by both isoforms was sensitive to citrate, with Mg2+ and Mg-citrate addition only formation of the ACC2 aggregates showed a dependence on citrate concentration. Mass spectrometry data indicated phosphorylation of Ser79 of ACC1 (a serine known to regulate activity), and the corresponding Ser221 of ACC2. Taken together, these data suggest that recombinant human ACC1 and ACC2 are differentially activated by citrate, most likely through conformational changes leading to aggregation, with ACC2 being more sensitive to this activator.
柠檬酸盐作为哺乳动物乙酰辅酶A羧化酶（ACC）的生理调节剂的作用已得到充分研究；然而，其机制尚未明确界定。在本研究中，我们发现柠檬酸盐使重组人ACC2的活性激活了约1000倍以上，但仅使重组人ACC1的活性激活了约4倍。这些数据最符合一个解释两个柠檬酸盐分子协同结合的模型。柠檬酸盐在较低浓度下激活ACC，在较高浓度下抑制ACC，ACC1和ACC2的表观解离常数（K(d)）分别为0.8±0.3和3.4±0.6 mM，表观抑制常数（K(i)）分别为20±8和38±8 mM。在不添加柠檬酸盐的情况下，抗生物素蛋白能迅速且完全地使ACC1和ACC2失活。添加10 mM柠檬酸盐可保护ACC2不被抗生物素蛋白失活；然而，柠檬酸盐对ACC1的保护作用不那么明显。通过动态光散射也观察到，在柠檬酸盐处理后，两种同工型呈现出不同的聚集模式。尽管两种同工型形成聚集体都对柠檬酸盐敏感，但添加Mg2+和柠檬酸镁时，只有ACC2聚集体的形成显示出对柠檬酸盐浓度的依赖性。质谱数据表明ACC1的Ser79（一个已知调节活性的丝氨酸）和ACC2相应的Ser221发生了磷酸化。综上所述，这些数据表明重组人ACC1和ACC2对柠檬酸盐的激活作用存在差异，很可能是通过导致聚集的构象变化，其中ACC2对这种激活剂更为敏感。
